This proposal explores the structure-function of the connexin channel by the use of pore blockers, focusing on two connexins in which defects cause neurological pathologies. Connexin protein forms gap junction channels, which are pathways for direct movement between cells of ions and cytoplasmic molecules, including most known second messengers. Serious pathologies arise from connexin defects, including demyelination, deafness, skin disorders and cataracts, depending on the connexin isoform affected. Despite acute biological and medical interest, the mechanism of the defining property of connexin channels - the ability of the pore to mediate selective molecular permeability between cells - has not been elucidated. Investigation of the structure and function of connexin pores has been hampered by the absence of molecular reagents that enter and bind in the pore. This class of reagents ("pore blockers") has been of inestimable value in elucidation of the structure-function of permeation of other channels. This project applies newly identified connexin pore blockers to investigate the connexin pore, and to thus obtain key information that has been long desired. Preliminary studies have identified two classes of carbohydrate-based connexin pore blockers, and established their feasibility as investigational tools of connexin channels. For the first class, novel glycinamide derivatives of aminobenzoic acid glycinamides (ABGs) were designed and conjugated to a size-indexed set of maltosaccharides. The resulting ABG-glycoconjugates act as reversible, high affinity blockers of molecular permeation through connexin pores in a size- and connexin-specific manner, whereas the maltosaccharides or the ABGs alone do not block. The second class of blockers are cyclodextrins (CDs), which are cyclized glucosaccharides. They also block connexin pores in a reversible, size-specific manner. For both classes of blockers, the correlation between the size of the molecule required for block and the relative width of the pore (determined using a size-indexed series of permeable sugars) indicate that their site of action is within the pore. The proposed studies build on this work. Aim 1 investigates the chemical determinants and mechanism of the intra-pore binding of the ABG- glycoconjugates. Aim 2 initiates application of the ABG-sugars to the study of connexin channels. Aim 3 utilizes naturally-occurring and modified CDs to probe the connexin pore. The projects primarily utilize a well- characterized reconstitution system to study heterologously-expressed connexin channels to obtain information unavailable by other means. It is anticipated that the development and application of these pore blockers will enable and inform the biophysical and cellular studies required to define the molecular mechanisms of intercellular communication in development and disease. In the present proposal, this analysis will be applied to Cx32 and Cx26, defects in which cause X-linked Charcot-Marie-Tooth disease neuropathy and sensorineural deafness, respectively.